EXPRESSION AND PURIFICATION OF ENZYMATICALLY ACTIVE RECOMBINANT GRANZYME-B IN A BACULOVIRUS SYSTEM

Granzyme B (GranB), a serine protease stored in the granules of cytoto xic T lymphocytes and natural killer cells, can initiate target cell a poptosis. To produce large amounts of purified active enzyme, recombin ant murine granzyme B (rGranB) was expressed from baculovirus in insec t cells. The expressed rGranB is secreted into the culture medium and can be readily purified to homogeneity by one-step affinity chromatogr aphy to yield 1.5 mg enzyme per liter insect cell medium. RGranB is re cognized by a GranB-specific anti-peptide antibody and is active again st synthetic substrate Boc-Ala-Ala-Asp-SBzl with kinetic constant (k(c at)/K-m 45,000 M(-1)s(-1)) comparable to purified human GranB. RGranB processes the caspase pro-CPP32 into its enzymatically active form and induces DNA fragmentation in isolated nuclei in the presence of cytos olic factors. The ability to express enzymatically active rGranB using the baculovirus system will help elucidate the role of this granzyme in the immune response. (C) 1998 Academic Press.